The present invention relates generally to plant genetic engineering, and specifically to both a novel gene whose polypeptide functions as a receptor of brassinolide and is useful for producing genetically engineered plants characterized as having a phenotype of increased crop yield, enhanced disease resistance and longer-lived vegetative growth phase and to the receptor function of Brassinosteroid 1 (BIN1) plasma membrane receptor.
The brassinosteroids are a unique class of biologically active natural products that possess plant steroidal hormone activity. Their low effective concentrations for use on crops make them environmentally safe and those brassinosteroids used on a large scale are generally non-toxic. At the physiological level, brassinosteroids elicit many changes and could represent a new class of hormones in plants. The economic aspects of the brassinosteroids may have worldwide effects. For example, the brassinosteroids can be used as plant protectants from both pesticide and environmental adversity. In addition, brassinosteroids appear to be useful for insect control. Further, brassinosteroids may regulate some stage of the reproductive cycle in plants, thereby providing the means to increase or decrease the reproductive process. For example, in certain horticultural crops, it may be desirable to eliminate the flowering process to ensure continuous production of other tissues such as leaves, bulbs and other storage organs. This modulation of the reproductive process could be important in the control of certain seed bearing weeds, where cessation of the flowering cycle eliminates future generations. Brassinosteroids also appear to stimulate root growth, and external application causes no deformity of plants.
Brassinosteroids qualify for classification as biochemical pesticides. Such pesticides are generally distinguished from conventional chemical pesticides by their unique modes of action, low effective concentration, target species, and specificity. Historically, the brassinosteroids have not been used in actual agricultural applications due to the expense involved in producing them as well as the difficulty in purifying them.
It is known that once hormones, such as glucocorticoid, enter a cell, they bind to specific receptor proteins, thereby creating a ligand/receptor complex. The binding of the hormone to the receptor is believed to initiate an allosteric alteration of the receptor protein. As a result, it is believed that the ligand/receptor complex is capable of binding with high affinity to certain specific sites on the chromatin nucleic acid. Such sites, which are known as response elements, modulate expression of nearby target gene promoters.
Recent evidence indicates that in addition to intracellular, genomic effects, steroids also exhibit non-genomic effects, ie., they affect the surface of cells and alter ion permeability, as well as release of neurohormones and neurotransmitters. Steroids such as estrogens and adrenal steroids and their naturally produced and synthetic analogs have shown membrane effects. In view of the foregoing, it appears that steroids may cause synergistic interactions between non-genomic and genomic responses resulting in alterations in neural activity or certain aspects of oocyte and spermatozoa maturation, for example.
Most multicellular organisms use steroids as signalling molecules for physiological and developmental regulation. Two different modes of steroid actions have been described in animal systems: the well-studied gene regulation response mediated by nuclear receptors, and the rapid non-genomic responses mediated by proposed membrane-bound receptors. See Beato, M., Herrlich, P. and Schutz, G. Steroid hormone receptors: many actors in search of a plot. Cell 83, 851-7 (1995); Mangelsdorf, D. J. et al. The nuclear receptor superfamily: The second decade. Cell 83, 835-839 (1995); Wehling, M. Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365-393 (1997); and Schmidt, B. M., Gerdes, D., Feuring, M., Falkenstein, E., Christ, M., Wehling, M. Rapid, nongenomic steroid actions: A new age? Front Neuroendocrinol 21, 57-94 (2000). Plant genomes do not appear to encode members of the nuclear receptor superfamily. For these reasons, it would be important to identify any new brassinosteriod receptors.
Although steroid hormones are important for animal development, the physiological role of plant steroids is largely unknown. The present invention is based on the discovery of the BIN1 gene, which encodes a polypeptide that functions as a receptor kinase which binds with brassinosteroids.
In one embodiment, the invention provides a trans-membrane receptor kinase, BIN1 polypeptide, comprising a plant steroid receptor of brassinsteroids. This BIN1 polypeptide has an active binding region having a 70 amino acid island region as an extracellular domain receptor. The 70 amino acid island region is required for brassinosteriod binding to the receptor on the cell membrane.
In another embodiment, the invention provides a BIN1 polypeptide wherein the polypeptide function in the brassinolide response pathway; has brassinolide-binding activity; an extracellular location of its functional binding site; brassinolide-binding activity which co-immunoprecipitates with BIN1; and trans-membrane receptor kinase activity that transduces steroid signals across the plasma membrane.
In yet another embodiment, the invention provides a BIN1 receptor kinase that has binding affinity of approximately Kd=7.4xc2x10.9 nM to 10.8xc2x13.2 nM depending on the number of BIN1 binding sites BMAX=2.66 pmole mgxe2x88x921 membrane protein. The BIN1 immunoprecipitated binding activity has a similar disassociation constant (Kd=15.2xc2x15 nM) as determined for membrane fractions.
Still another embodiment of the invention provides a BIN1 mutant with missense mutations in the kinase domain (BIN1-104, A1031T) or in a region of the extracellular domain near the transmembrane domain (BIN1-102, T750-I) wherein the brassinolide binding activity is similar to the wild type and the biosynthetic mutant det2. Another BIN1 mutant with missense mutations (BIN1-6, G644-D) and a mutation causes a premature translation step (BIN1-116, Q583-stop), both in the 7 amino acid island region, wherein the brassinolide binding activity is greatly reduced.